Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR–RFLP

Background: This study was carried out to identify Theileria spp. infections in goats and ticksin Kermanshah Province, western Iran from May–Sep 2015. Methods: For differentiation of different Theileria spp. both blood and tick samples were examined by nested PCR-RFLP. Results: Light microscopy of blood smears revealed Theileria spp. infection in 22 (5.5%), while 68 (17%) of blood samples were positive using nested PCR. Out of 68 positive samples, 85.3% (58/68) and 11.7% (8/68) were respectively positive for Theileria ovis and T. lestoquardi. Mixed infection was detected in 3% (2/68) cases. Overall, 420 ixodid ticks belong to seven different hard ticks species were collected from goats. Rhipicephalus turanicus 112 (26.7%), R. sanguineus 95 (22.6%), R. bursa, 91(21.7%), Hyalomma anatolicum, 55(13.1%), H. excavatum 27(6.4%), H. marginatum, 22(5.3%) and Dermacentor marginatus, 18(4.2%) were the main tick species infesting goats. The PCR products obtained from ticks were subjected to the differentiation of Theileria species. Respectively, 2 and 8 pools of H. marginatum and R. turanicus salivary glands were infected with T. ovis and T. lestoquardi. In addition, T. annulata and T. lestoquardi infection weredetected in three pools of H. anatolicum. Conclusion: This is the first report of goats and collected ticks to Theileria spp infection in Iran. The results suggest that T. ovis has a higher prevalence than T. lestoquardi. It is also postulated H. marginatum, R. turanicus and H. anatolicum might play an important role in the field as a vector of Theileria spp in this area.

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Molecular surveillance of Theileria ovis, Theileria lestoquardi and Theileria annulata infection in sheep and ixodid ticks in Iran

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v80i1.635 ◽

2013 ◽

Vol 80 (1) ◽

Cited By ~ 9

Author(s):

Gholamreza Razmi ◽

Saeed Yaghfoori

Keyword(s):

Polymerase Chain Reaction ◽

Salivary Glands ◽

Nested Pcr ◽

Ixodid Ticks ◽

Chain Reaction ◽

Blood Samples ◽

Theileria Lestoquardi ◽

Blood Smears ◽

Pcr Rflp ◽

Polymerase Chain

A molecular study was undertaken to detect Theileria ovis, Theileria lestoquardi and Theileria annulatain sheep and tick vectors. Investigation was conducted from 2010 to 2011 in the south of Khorasan Razavi Province, Iran. A total of 150 blood samples were collected from 30 different sheep flocks. In addition, ixodid ticks were sampled from the same flocks. The stained blood smears were microscopically examined for the presence of piroplasms and a semi-nested polymerase chain reaction-restriction (PCR) was used for subsequent molecular speciation. Salivary glands were isolated from the ticks and subsequently analysed by semi-nested PCR. polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to differentiate between T. lestoquardi and T. annulata from PCR-positive samples. Theileria species infection was microscopically detected in 18.6% of blood smears. The presence of T. ovis and T. lestoquardi or T. annulata was detected by semi-nested PCR in 58.6% and 6.6% of blood samples respectively. In total, 169 ixodid ticks were collected from different areas of the province. The most prevalent ticks were Rhipicephalus turanicus (n = 155; 91.7% of the total), followed by Hyalomma anatolicum anatolicum (n = 8; 4.7%) and Hyalomma marginatum turanicum (n = 6; 3.5%). From an organ pooling of 33 ticks, three pools of salivary glands from R. turanicus were positive for Theileria species by semi-nested PCR. Of the three R. turanicus samples testing positive for Theileria species, two (6.1%) were positive for T. ovis and one (3.0%) for T. lestoquardi or T. annulata. Amongst the 11 PCR-positive samples for T. lestoquardi or T. annulata, 10 were positive for T. lestoquardi and one sample was positive for both T. lestoquardi and T. annulata using PCR-RFLP. The results also demonstrated that PCR-RFLP could be used for the detection of T. ovis. Based on the results, it can be concluded that T. ovis has a higher prevalence than T. lestoquardi, and that R. turanicus could be a possible vector for T. ovis and T. lestoquardi. Finally, the PCR-RFLP based on Msp1 restriction enzyme is a simple method for differentiation of Theileria species in sheep and ixodid ticks.

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Identification of individual DNA molecule of Mycobacterium tuberculosis by nested PCR-RFLP and capillary electrophoresis

Talanta ◽

10.1016/j.talanta.2008.06.004 ◽

2008 ◽

Vol 77 (1) ◽

pp. 182-188 ◽

Cited By ~ 9

Author(s):

Po-Ling Chang ◽

Wen-Shyang Hsieh ◽

Chia-Lien Chiang ◽

Belinda Yen-Liberman ◽

Gary W. Procop ◽

...

Keyword(s):

Capillary Electrophoresis ◽

Mycobacterium Tuberculosis ◽

Nested Pcr ◽

Dna Molecule ◽

Pcr Rflp

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Sanitary Status of the Grapevine Germplasm Collection in Republic of Srpska

Агрознање ◽

10.7251/agren1602143d ◽

2017 ◽

Vol 17 (2) ◽

pp. 143

Author(s):

Duška Delić ◽

Biljana Lolić ◽

Gordana Đurić ◽

Tatjana Jovanović-Cvetković

Keyword(s):

Nested Pcr ◽

Germplasm Collection ◽

Elisa Test ◽

Grapevine Fanleaf Virus ◽

Pcr Rflp ◽

Near Future ◽

Infected Grapevine ◽

Rflp Assay ◽

Das Elisa

In July 2015, 179 grapevine plants belonging to 16 grapevine autochthonous cultivars were assessed for sanitary status using DAS ELISA test for the presence of: Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2)and Grapevine leafroll-associated virus 3 (GLRaV-3). Furthermore, surveyfor the phytoplasma presence and laboratory analyses using nested-PCR/RFLP assay was conducted at the beginning of September 2015 on grapevine cultivars which were not positive in DAS ELISA test for the presence of the four viruses. Out of 179 tested plants with DAS ELISA test, 146 (81%) were positive for the presence of at least one virus. The most widespread viruses were GFLaV- 1 and GFLaV- 3 with approximately 80 % of grapevines infected. Nested–PCR/RFLP assay showed that out of 33 tested samples 2 were positive for the presence of phytoplasmas from 16SrXII group. Sanitation of infected grapevine cultivars is needed in near future.

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Assessment of Giardia and Cryptosporidium Assemblages/ Species and Their Viability in Potable Tap Water in Beni-Suef, Egypt Using Nested PCR/RFLP and Staining

Iranian Journal of Parasitology ◽

10.18502/ijpa.v14i3.1475 ◽

2019 ◽

Author(s):

Doaa HAMDY ◽

Ayman El-BADRY ◽

Wegdan ABD EL WAHAB

Keyword(s):

Water Samples ◽

Rural Areas ◽

Nested Pcr ◽

Membrane Filtration ◽

Tap Water ◽

Significant Risk ◽

Waterborne Diseases ◽

Filtration Method ◽

Pcr Rflp ◽

Blue Stain

Background: The protozoan Giardia and Cryptosporidium are responsible for most water-borne diseases all over the world. The extent and number of outbreaks of waterborne diseases suggests a significant risk of their potential transmission via drinking water. This study aimed to document the prevalence and viability of Giardia and Cryptosporidium (oo) cysts in tap water samples in Beni-Suef Governorate, Egypt and to detect the predominant Giardia and Cryptosporidium assemblages/species using nested PCR/ Restriction Fragment Length Polymorphism (RFLP) confirmed by further sequencing of positive samples. Methods: A total of 80 tap water samples were collected throughout a year from four big centers and filtered using the membrane filtration method. Samples were stained by Lugol’s iodine, Modified Zeihl-Neelsen (MZN) (to detect prevalence) and trypan blue stain (to detect viability). Nested PCR-RFLP and sequencing were used for molecular characterizations and genotyping of the detected Giardia and Cryptosporidium. Results: Giardia and Cryptosporidium DNA was detected in 20 (25%) and 29 (36.3%) samples respectively, with predominance of Giardia assemblage B (85%) and C. hominis (75.9%). The prevalence and viability of both parasites (oo) cysts showed seasonality which peaked in summer and were greater in Beba center and in rural areas. Conclusion: To our knowledge, no studies have been done in these areas before. The anthroponotic transmission has an important role in giardiasis and cryptosporidiosis epidemiology in this studied area.

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Molecular Characterization ofCryptosporidiumspp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

Journal of Tropical Medicine ◽

10.1155/2016/6834206 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6 ◽

Cited By ~ 12

Author(s):

Jasem Saki ◽

Masoud Foroutan-Rad ◽

Reza Asadpouri

Keyword(s):

Molecular Characterization ◽

Nested Pcr ◽

18S Rrna ◽

Restriction Enzymes ◽

18S Rrna Gene ◽

Rrna Gene ◽

Wild Rodents ◽

Nested Polymerase Chain Reaction ◽

Pcr Rflp ◽

Southwest Of Iran

Background. Rodents could act as reservoir forCryptosporidiumspp. speciallyC. parvum, a zoonotic agent responsible for human infections. Since there is no information aboutCryptosporidiuminfection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization ofCryptosporidiumspp. in this region.Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method usingSspIandVspIrestriction enzymes was carried out to genotype the species and then obtained results were sequenced.Results. Three out of 100 samples were diagnosed as positive and overall prevalence ofCryptosporidiumspp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples andC. parvumpattern was identified. Finally PCR-RFLP findings were sequenced and presence ofC. parvumwas confirmed again.Conclusions. Our study showed rodents could be potential reservoir forC. parvum. So an integrated program for control and combat with them should be adopted and continued.

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